The major goal of the research supported by this grant is to define the biochemical and structural rules that determine how protein kinases identify their substrates and to understand how phosphorylation of proteins leads to the assembly of signaling complexes via phospho-protein binding domains. Specifically, we are interested in defining the protein kinase signaling networks that control cell growth and cell survival. With support from this grant over the past decade we have developed oriented peptide library approaches that have begun to explain how protein kinases phosphorylate specific substrates, on the basis of the sequence context of the phosphorylation site. In addition, with support from this grant we have developed a novel technology that has uncovered new and unexpected phosphoprotein-binding domains, including the polobox domain of polo-like kinases, the C2 domain of Protein Kinase C delta and Protein Kinase C theta and, surprisingly, the regulatory domain of the M2 isoform of pyruvate kinase. During the next granting period we intend to use these and other approaches to identify additional protein kinases and phospho-protein binding domains that play key roles in cell growth and survival pathways and fit them into existing signaling networks. The sequence selectivity matrices that are derived from our peptide library studies will be uploaded into our Scansite program, which facilitates the identification of substrates of kinases from screens of protein and phosphoprotein sequence databases. Finally we will attempt to define the structural basis for the phosphoprotein-binding interactions that we identify. These studies will help flesh out protein kinase signaling networks and suggest new nodes for pharmaceutical intervention to treat cancers and other diseases that involve dysregulated cell growth. PUBLIC HEALTH RELEVANCE: Cancer cells differ from cells in normal adult tissues in that they continue to grow indefinitely, resulting in tumors. Protein kinase signaling networks control the growth of cancer cells and drugs that target some of these kinases have been approved for treating cancers. The focus of this grant is to understand the intracellular protein kinase signaling networks that control the growth of normal and cancerous cells and identify new targets for pharmaceutical intervention.